The regulation of sporulation and its interplay with the complex cellular processes that control growth and division in Bacillus species has been compromised in Bacillus anthracis by the virulence plasmids pXO1 and pXO2 with their own regulatory agenda. The research we have accomplished and propose here in B. anthracis is dedicated to unraveling the complications that their presence generates in the basic molecular interactions that drive cell growth and development. Anthrax toxin expression represents the ultimate end product of the interaction and coordination of the cellular and gene specific regulatory circuits. We have developed transposon vectors to probe the genes affecting the regulatory circuits. Transposon insertion strains will be isolated using specific reporters that assess the transcriptional expression of the genes encoding the protective antigen, and its major known regulators, AtxA and PagR. Our studies indicate that post-translational regulation of the activity of AtxA occurs by phosphorylation through the PTS sugar transport system. The specific components of this system responsible for AtxA regulation will be identified by mutagenesis experiments. The location of the phosphorylated residues on AtxA will be determined. The virulence plasmids were found to encode genes structurally similar to the sensor domain of a major sporulation sensor kinase and the expression of these genes was shown to inhibit that kinase and sporulation. Studies will be undertaken to identify the signal ligand bound to these proteins that allows the plasmids to affect the cellular circuits for sporulation. Structural studies that visualized the protein folds of the plasmid sensor domains and that of the PagR protein will be continued and focused on the DNA-bound structure of PagR and sporulation sensor kinases. Alanine scanning mutagenesis will be used to probe for possible effectors of PagR activity. A markerless deletion program on sporulation sensor kinases will be undertaken to determine those kinases responsible for allowing toxin synthesis during growth. This program has the goal of generating knowledge of the pathogenic regulatory mechanisms employed by toxin producing spore-forming bacteria for which little is known.